1. Field of the Invention
This invention relates to a novel use for a protein derived from keratin. In particular, it proposes the use of alpha-keratose as a blood plasma expander, for the preservation and transportation of organs and for the formed elements of blood.
2. Description of the Background Art
Heretofore, an intense research effort has been made and many materials have been developed that lend themselves for possible use as a blood plasma expander. A blood plasma expander is generally used for fluid replacement in the treatment of extensively burned individuals to remove burn toxins and/or prevent burn shock. Fluid replacement therapy may also be required by individuals with open wounds and extensive external and/or internal bleeding, immunologic defects, clotting problems, or severe diarrhea.
Blood plasma expanders have been made from materials consisting of crystalloid solutions or colloidal-like polymeric solutions. The crystalloid materials include saline, compositions of saline and glucose, Hartmann""s solution, and Ringer""s solution. The colloidal-like polymeric materials include pectin, gelatin, albumin, hydroxyethyl starch, polyvinyl pyrrolidone, dextran and cellulose derivatives.
Some blood plasma expanders have been made from blood and blood fractions. However, the use of blood and blood fractions is fraught with disadvantages. For example, the use of whole blood often is accompanied with the risk of transmission of hepatitis-producing viruses or other viruses which complicate the patient""s recovery. Additionally, the use of whole blood requires blood-typing and cross-matching to avoid immunohematological problems and interdonor incompatibility.
Therefore, a blood fraction plasma, which is a physiologically balanced colloidal solution that fulfills many of the requirements of a blood volume expander, cannot be safely used for this purpose unless further efforts in time and expense are made to remove infectious agents. It would also be advantageous to remove isoagglutinins. Clearly it is desirable to use non-blood based blood substitutes. In view of the above presentation, it becomes immediately apparent that a need exists for a therapeutic product useful as a blood plasma expander without the above drawbacks.
Plasma and albumin, as blood plasma expanders, have disadvantages in their availability, initial expense, storage, and risk of viral infection. Whereas, dextrans of high molecular weight are especially useful in that they are not excreted as rapidly as low molecular weight dextrans, they present the dangers of rouleaux formation, sludging of erythrocytes, and possible anaphylaxis. Hence the compromise using dual high and low molecular weights of oncotic agents with differential clearance rates, such as dextrans and hydroxyethyl starches (HES) to overcome this problem (U.S. Pat. No. 5,945,272).
Since experimental work with mice has shown the use of serum to be no more effective than its sodium content (Rosenthal; cited by Fox, 1967) and the use of colloids serves both to raise venous pressure and to reduce sodium excretion, it should be possible to devise a salt-colloid mixture suitable for fluid replacement therapy.
It would therefore be advantageous if the colloid portion of a blood plasma expander could be a non-antigenic aqueous drug and virus-free solution which could act as an oncotic agent, which could serve as food for various tissue cells, which could be used for cryopreservation of tissues preparatory to transplantation, which could be freely miscible with other electrolytes, nutrients, oncotics, and oxygen bearing substances known in the blood transfusion art and which could be sterilized and stored long term for use at ambient room temperatures.
Accordingly, there is a critical and continual need for a blood plasma expander that can be used for volume deficiency shock, as an alleviant in anaphylactic and allergic shock, for replacing plasma lost after burns and as a result of diarrhea and for preserving and transporting organs.
Accordingly, it becomes an object of the invention to provide a salt-colloid mixture suitable as a blood plasma expander for all blood types, which does not have the drawbacks of plasma/albumin (problems with donor accessibility, possible viral infection, foreign antigens, cold storage), or the balancing of high and low molecular weight solutions of dextrans or hydroxyethyl starches.
Another object of the invention is to provide a novel agent useful as a blood and plasma expander by dint of molecular size large enough to allow it to maintain intravascular residence and osmotic pressure for a prolonged time.
Yet another object of the invention is to provide a blood and plasma expander which can serve as a source of nutrient growth factors for the cells it bathes.
A further object of the invention is to provide a useful blood and plasma expander which is miscible and compatible with all components of blood and is itself non-toxic, non-antigenic, and non-pyrogenic.
Still a further object of the invention is to provide a blood substitute and plasma expander which is stable under prolonged storage and is either safely discharged from the body or degraded to nutrient components.
Another object of the invention is to provide a blood-free plasma expander and blood substitute for use in a subject in need thereof, comprising a solution of alpha-keratose.
Yet another object of the invention is to provide a pharmaceutical composition useful as a blood plasma expander and blood substitute comprising a therapeutically effective amount of alpha-keratose that is soluble in aqueous and physiological fluids.
Another object of the invention is to provide a pharmaceutically acceptable carrier comprising a solution of alpha-keratose.
Yet another object of the invention is to provide a buffer comprising a solution of alpha-keratose.
Still another object of the invention is to provide a method of treating a human in need of blood by intravenously administering to the human an effective amount of a blood substitute comprising alpha-keratose.
Still yet another object of the invention is to provide a method for increasing the volume of the blood circulatory system wherein the method comprises transfusing into a system having a decreased volume, a quantity of a blood volume expander which consists of a solution of alpha-keratose, wherein the quantity is transfused in an effective amount to increase said volume.
Another object of the invention is to provide a method for the treatment of shock which comprises administering into the circulatory system of a mammal in shock, a blood plasma expander consisting of a solution of alpha-keratose, in an effective amount to alleviate said shock.
Yet another object of the invention is to provide a method for maintaining an isolated mammalian organ in a viable state which method comprises perfusing the organ with an effective amount of a perfusate consisting of a solution of alpha-keratose.
Another object of the invention is to provide an oncotic agent which has been shown to be a complete substitute for fetal calf serum in tissue cultured cells which have been adapted to growth in peptone dialysate.
Still another object of the invention is to provide a method for removing pathogens, poisons or toxins from blood present in a patient, the method comprising the steps of infusing the patient with a sufficient volume of a blood substitute solution comprising at least one water soluble oncotic agent while removing an equal volume of blood from the patient to reduce the hematocrit of the patient to 10 to 15%; separating the blood cells of the blood from the pathogens, toxins or poisons; resuspending the blood cells in a blood plasma expander comprising a water soluble oncotic agent to produce a blood cell comprising solution; and infusing the patient with the blood cell comprising solution; whereby the pathogens, toxins or poisons are removed from the blood of the patient.
The foregoing has outlined some of the pertinent objects of the present invention. These objects should be construed to be merely illustrative of some more prominent features and applications of the intended invention. Many other beneficial results can be attained by applying the disclosed invention in a different manner or modifying the invention within the scope of the disclosure. Accordingly, other objects and a fuller understanding of the invention may be had by referring to the summary of the invention and the detailed description of the preferred embodiment in addition to the scope of the invention defined by the claims.
This invention is concerned with the use of soluble keratins as a blood plasma expander. In addition, the soluble keratins can be used as a vehicle for transportation and preservation of organs and the formed elements of blood.
A feature of the present invention is to provide a novel agent useful as a blood and plasma expander by dint of molecular size large enough to allow it to maintain intravascular residence and osmotic pressure for a prolonged time.
Yet another feature of the present invention is to provide a blood and plasma expander which can serve as a source of nutrient growth factors for the cells it bathes.
A further feature of the present invention is to provide a useful blood and plasma expander which is miscible and compatible with all components of blood and is itself non-toxic, non-antigenic, and non-pyrogenic.
Still a further feature of the present invention is to provide a blood substitute and plasma expander which is stable under prolonged storage and is either safely discharged from the body or degraded to nutrient components.
Another feature of the present invention is to provide a blood-free plasma expander and blood substitute for use in a subject in need thereof, comprising a solution of alpha-keratose.
Yet another feature of the present invention is to provide a pharmaceutical composition useful as a blood plasma expander and blood substitute comprising a therapeutically effective amount of alpha-keratose that is soluble in aqueous and physiological fluids.
Another feature of the present invention is to provide a pharmaceutically acceptable carrier comprising a solution of alpha-keratose.
Yet another feature of the present invention is to provide a buffer comprising a solution of alpha-keratose.
Still another feature of the present invention is to provide a method of treating a human in need of blood by intravenously administering to the human an effective amount of a blood substitute comprising alpha-keratose.
Still yet another feature of the present invention is to provide a method for increasing the volume of the blood circulatory system wherein the method comprises transfusing into a system having a decreased volume, a quantity of a blood volume expander which consists of a solution of alpha-keratose, wherein the quantity is transfused in an effective amount to increase said volume.
Another feature of the present invention is to provide a method for the treatment of shock which comprises administering into the circulatory system of a mammal in shock, a blood plasma expander consisting of a solution of alpha-keratose, in an effective amount to alleviate said shock.
Yet another feature of the present invention is to provide a method for maintaining an isolated mammalian organ in a viable state which method comprises perfusing the organ with an effective amount of a perfusate consisting of a solution of alpha-keratose.
Another feature of the present invention is to provide an oncotic agent which has been shown to be a complete substitute for fetal calf serum in tissue cultured cells which have been adapted to growth in peptone dialysate.
Still another feature of the present invention is to provide a method for removing pathogens, poisons or toxins from blood present in a patient, the method comprising the steps of infusing the patient with a sufficient volume of a blood substitute solution comprising at least one water soluble oncotic agent while removing an equal volume of blood from the patient to reduce the hematocrit of the patient to 10 to 15%; separating the blood cells of the blood from the pathogens, toxins or poisons; resuspending the blood cells in a blood plasma expander comprising a water soluble oncotic agent to produce a blood cell comprising solution; and infusing the patient with the blood cell comprising solution; whereby the pathogens, toxins or poisons are removed from the blood of the patient.
The foregoing has outlined rather broadly the more pertinent and important features of the present invention in order that the detailed description of the invention that follows may be better understood so that the present contribution to the art can be more fully appreciated. Additional features of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.
Keratins are unique to the animal kingdom. Keratins are complex proteins found throughout the tissues of the body most notably in skin, hair, nail, claw, horn, hoof, fur, wool, quill, and feather.
The colloid portion, or part of the colloid portion of the solution of the present invention, is a non-antigenic, nutrient, soluble fraction of keratin.
Keratin derivatives are chemically and immunologically distinct according to the species from which derived, and according to the method of extraction and/or solubilization. For example, keratin derivatives can be extracted and/or solubilized by reduction by thiol groups and solution in alkali, oxidation, sulfitolysis and solution in urea, oxidative sulfitolysis or enzymatic cleavage (Crewther et al., 1965). Such soluble keratin derivatives may be sized by column chromatography, degraded to smaller fragments, recombined in novel ways, or otherwise manipulated, filter sterilized, and stored at room temperature, obviating both cold storage and viral threat.
The present invention embodies the soluble alpha-keratose fraction of insoluble alpha-keratin; i.e., the fibrous protein microfibrils in the form of intertwined filamentous alpha-helices found in native keratins. The preferred source of material is human hair (abundant, inexpensive, renewable, and non-antigenic) in which the hair cortex is packed with alpha-keratin.
The preparation of alpha-keratose from human hair (Widra, 1966) or wool xe2x80x9ctopsxe2x80x9d(Widra 1989) was developed as an integral and essential component of a series of biodegradable hydrophilic wound covers, as described by Widra (1986). The laboratory preparation method is repeated here, wherein the first five steps may be conveniently performed in a single one liter Erlenmeyer flask:
1. Twelve grams of human hair is degreased with ether-alcohol (1:1) and then washed in water.
2. 320 ml of water and 80 ml of concentrated (40%) peracetic acid is added to the hair.
3. The hair-acid mixture is refrigerated at 4xc2x0 C. with constant stirring for 24 hours.
4. The hair is then freed of the peracetic acid by decantation and thorough washing with separate water rinses.
5. The washed hair is then covered with 800 ml of 3N ammonium hydroxide, refrigerated, and stirred for 24 hours at a temperature of 4xc2x0 C.
6. The total soluble protein (TP) fraction is then cleared of solids by centrifugation and coarse filtration to remove any visible particulates.
7. The TP fraction is dialyzed through cellulose tubing (14,000 molecular weight cut off) against water.
8. Alpha-keratose is precipitated from the dialyzed TP fraction with small incremental additions of 0.1 N hydrochloric acid, stirring between additions. This procedure is continued until no further precipitate forms.
9. The white precipitate is collected by centrifugation and the supernatant gamma-keratose is discarded.
10. The alpha-keratose precipitate is washed in water, recentrifuged, then resolubilized in 0.1 N ammonium hydroxide.
11. A second cycle of precipitation, washing, and solubilization is run on the alpha-keratose before final dialysis against water, microfiltration (22 micra), and storage in a sterile container. (The alpha-keratose solution may be concentrated by simple pervaporation through the dialysis bag, then assayed for protein content.)
The resulting light amber solution of alpha-keratose is faintly positive to Nessler""s solution and just on the alkaline side of neutrality (pH 7.1-7.2).
The average molecular weight of the alpha-keratose fraction of human hair determined by ultracentrifugation is 45,000 daltons (Widra, 1989).
Alpha-keratose from human hair (15 ml, 5 mg per ml) failed to raise antibodies in a rabbit, whereas gamma keratose did so, cross reacting with both TP and alpha-keratose (Widra, 1966). The procedures freeing and fragmenting the linear alpha-keratin fibrils from matrix (gamma keratose) proteins may also destroy antigenicity. Alpha-keratose, itself, may be resolved into 3 subfractions by disc electrophoresis on acrylamide gel (Rhodes et al, 1967). The main subfraction is characteristic of serum albumin in its speedy migration to the anode, isoelectric point (pH 4.8), high sulfur content, and paucity of tryptophane residues (Peters, 1970; O""Donnell and Thompson, 1961; He and Carter, 1992).
The total soluble protein (TP) or the alpha-keratose derived from human hair is non-toxic to cells in tissue culture using primary human embryonic kidney cells, a Wistar Institute (Philadelphia, Pa.) strain of diploid human embryonic lung cells (WI38), African green monkey kidney cells, and heteroploid rabbit heart cells. Furthermore, in such cells adapted to growth in Bacto-peptone supplement as a substitute for fetal bovine serum. (FBS) (Pumper et al 1965; Taylor et al 1972), alpha-keratose could completely replace the peptone and/or FBS as a nutrient growth factor (Widra and Pumper 1981).
Since alpha-keratose is miscible in all proportions with the usual physiologic salt/electrolyte crystalloid solutions used in transfusion therapy, this novel oncotic and nutrient agent may be used to form a salt-colloid mixture suitable for fluid replacement therapy.
The blood plasma expander of this invention is an aqueous solution without any of the cells normally found in whole blood or blood plasma and, as such, can generally be safely stored for much longer periods than whole blood or blood plasma and can be used without cross-matching or haplotyping between donor and recipient. Since the blood plasma expander solutions of this invention are totally synthetically produced there is substantially no risk of blood contaminants, such as bacterial, viral or other blood contaminants being introduced into the solutions. This is especially important in this age of hepatitis, AIDS and other blood transmitted diseases.
In greater detail, the present invention comprises a mixture of components which when placed in aqueous solution may be used to expand the blood plasma volume of a subject in need thereof. The forgoing components may be provided as a dry sterile mixture to which sterile diluent such as water, saline solution or dextrose solution may be added to form an aqueous solution. If provided as a dry sterile mixture, the materials may be provided in a sterile container suitable for mixture with sterile diluent such as sterile water, sterile saline, sterile dextrose solution, or a suitable commercially available electrolyte solution. Alternatively the mixture of materials may be provided in a sterile container as an aqueous solution.
In the following examples, production was scaled up and powdered alpha-keratose was manufactured as follows:
1. 76.7 g of human hair were swirled in ethyl ether and then washed with distilled water (dH2O).
2. 1000 ml of dH2O and 250 ml peracetic acid were added.
3. Solution was refrigerated at 4xc2x0 C. overnight with occasional swirling.
4. The solution was decanted to remove the peracetic acid and the solids washed with 3 liters of dH2O on a Buchner funnel.
5. The washed solids (hair) were immersed in 2.5 liters of 3N ammonium hydroxide and stirred at 4xc2x0 C. overnight.
6. The solids were removed by centrifugation at 5000xc3x97G for 15 minutes.
7. The supernate was then clarified through Whatman 54 paper by vacuum.
8. The solution was dialyzed overnight against flowing dH2O.
9. Material was precipitated by incremental addition of 0.1N HCl until no more precipitate was observed to form.
10. The precipitate was collected by vacuum filtration on Whatman 54 paper and washed with dH2O.
11. The precipitate was resolubilized in 0.3N ammonium hydroxide.
12. Solution was dialyzed against flowing dH2O overnight.
13. Solution was removed from dialysis and filtered through Millipack 200 (0.22 micra) into a depyrogenated tray for lyophilization.
14. Material was lyophilized and collected in a class 10,000 area, transferred to a biological safety cabinet (class 100), then stored in sterile containers.
This totally soluble, sterile, dry material gives xe2x80x9coff-the-shelfxe2x80x9d convenience and flexibility in preparing solutions with different concentrations of alpha-keratose.